effective design of multiplexed quantitative srm assays with skyline
DESCRIPTION
Effective design of multiplexed quantitative SRM assays with Skyline. Joseph Brown, Ph.D. Pacific Northwest National Laboratories. Minneapolis, MN; Skyline Workshop; 61 st ASMS. SRM pipeline flowchart. Investing on assay development simplifies data analysis. - PowerPoint PPT PresentationTRANSCRIPT
Effective design of multiplexed quantitative SRM assays with Skyline
Joseph Brown, Ph.D.Pacific Northwest National LaboratoriesMinneapolis, MN; Skyline Workshop; 61st ASMS
SRM pipeline flowchart
Investing on assay development simplifies data analysis
How do you choose the “best” transition, or is an “OK” transition sufficient?
Pepti
des
Tran
sitio
ns
• Prior Knowledge• Previous Experiments• Public Databases
• NIST• PeptideAtlas• GPM
• Prediction Software• SRM Collider
SRM Collider to predict potential matrix interferences
Assessing matrix interference
Heavy
Light
Remove b6-ion
Interfering Peak
Spectral libraries visualization through Skyline
Transition scheduling
Transition scheduling
Collision energy optimizationPrecursor Product ion Ramping CE504.2363 613.3043 11.3504.2363 613.3043 12.3504.2363 613.3043 13.3504.2363 613.3043 14.3504.2363 613.3043 15.3504.2363 613.3043 16.3504.2363 613.3043 17.3504.2363 613.3043 18.3504.2363 613.3043 19.3504.2363 613.3043 20.3504.2363 613.3043 21.3
RT: 15.90 - 44.01
16 18 20 22 24 26 28 30 32 34 36 38 40 42 44Time (min)
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20.98
21.34
19.0618.65
21.39
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25.6923.88
20.53 26.8521.9228.91
37.4542.6640.42
33.5531.0818.57 42.0839.0736.30
NL:2.28E7TIC MS CPTAC_SRM_Batch1_1_80min_CEramping_19Ap13_Legolas_W22511A2
CPTAC_SRM_Batch1_1_80min_CEramping_19Ap13_Legolas_W22511A2 #588 RT: 20.92 AV: 1 NL: 6.21E5T: + c NSI SRM ms2 683.390 [584.252-584.254, 584.262-584.264, 584.272-584.274, 584.282-584.284, 584.292-584.294, 584.302-58 ...
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A Composite Scan 3 transitions / 11 CEs
-5
-4-3
-2
-1
0+1
+2+3
+4
+5
-5
-4
-3-2-1
0
+1
+2
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+5
-5-4
-3-2-10
+1+2
+3+4
Precursor Product ion Ramping CE504.2363 243.8715 11.3504.2363 243.8715 12.3504.2363 243.8715 13.3504.2363 243.8715 14.3504.2363 243.8715 15.3504.2363 243.8715 16.3504.2363 243.8715 17.3504.2363 243.8715 18.3504.2363 243.8715 19.3504.2363 243.8715 20.3504.2363 243.8715 21.3
Precursor Product ion Ramping CE504.2363 874.3346 11.3504.2363 874.3346 12.3504.2363 874.3346 13.3504.2363 874.3346 14.3504.2363 874.3346 15.3504.2363 874.3346 16.3504.2363 874.3346 17.3504.2363 874.3346 18.3504.2363 874.3346 19.3504.2363 874.3346 20.3504.2363 874.3346 21.3
Skyline automates collision energy optimization
Peptide distinguishes T1D from healthy control
Platelet basic protein abundance in human serum
Zhang et al. 2013 J Exp Med.
Increased fractionation with PRISM-SRM facilitates deep-dive approach
Shi et al. 2012 PNAS
Time (min)
QQQ MS
Q1 Q2 Q3
QQQ MS
Q1 Q2 Q3
SRM Signal
Time (min)
3 µm, C18 column(200 µm i.d. × 50 cm)
1.7 µm, C18 column(75 µm i.d. × 25 cm)
iSelection
96 well plate
Conventional SRM analysis
On-line monitoring heavy peptides
Capillary LC (high pH)
Nano LC (low pH)
#2 #6 #10
Fraction multiplexing
High-pressure, high-resolution separations coupled with intelligent selection & multiplexing
Use Skyline to visualize and inspect fractions
PRISM-SRMConventional SRMITTRPDLPYEPPR
Increased S/N
Reduced Interference
Detection of an ERG peptide in TMPRSS2-ERG positive vs negative prostate cancer cell lines
VIVPADPTLWSTDHVR
(+) (+) (-) (-) (-) (-)
N.D. N.D. N.D. N.D.
Acknowledgments
Tao LiuWeijun QianTujin ShiTom FillmoreQibin ZhangChaochao WuJintang HeYuqian GaoTom MetzSam PayneGordon AndersonDavid CampKarin RodlandDick Smith
T1D-specific peptide
1.68.5
Zhang et al. 2013 J Exp Med.
Pro-platelet basic protein100 HC, 50 T1D 10 HC, 10 T1D 50 T2D